The genomic contribution to the development of very early onset inflammatory bowel disease (VEO-IBD), or IBD diagnosed at <6 years of age, remains understudied, yet elucidation of genetic risk factors would undoubtedly enhance our understanding of pathogenesis and suggest novel therapeutic approaches. Patients with VEO-IBD often present with greater extent and severity of disease than older onset IBD. Traditional therapies with immunosuppression often fail and in cases of immune deficiencies, are inappropriate. The aggressive phenotype, early onset, and strong family history points to an enrichment of monogenic defects in VEO-IBD. Indeed, we and others have identified causal genetic variants in VEO-IBD. Whole exome sequencing (WES) has radically changed our approach to VEO-IBD. However, linking the WES identified variant to the development of the VEO-IBD phenotype remains difficult. The goal of this proposal is to expand the repertoire of highly penetrant, causal variants responsible for VEO-IBD through a large scale sequencing study of a well characterized patient cohort, followed by replication in independent cohorts and functional validation of some of the identified variants. Our central hypothesis is that single gene defects are responsible for a large proportion of cases with VEO-IBD, and they can be effectively characterized by a multidisciplinary approach starting from state-of-the-arts genomic studies. In addition, we will test whether the identified VEO-IBD genes and pathways are specific to early onset cases or are also involved in susceptibility to the more common form of older onset IBD. Finally, we will specifically test the hypothesis that causal variants in specific genes and pathways will be associated with changes in the gut microbiome composition that will support their role in VEO-IBD. The rapidly increasing incidence of VEO-IBD cannot be explained by genetic susceptibility alone, and suggests a role of the gut microbiota as a driver of disease. To test our central hypothesis, in Specific Aim 1 we will create a dataset of potential causal mutations in VEO-IBD patients by WES. Our preliminary data using WES in VEO-IBD has already identified causal variants confirming the enrichment of monogenic defects in VEO-IBD. We will now increase our sample size to create a large catalogue of candidate genes that we will validate in additional independent VEO-IBD cases, and test for association with older onset IBD. In Specific Aim 2, candidate VEO- IBD variants will be subject to functional studies to confirm their causal effects. Finally, in Specific Aim 3, we will correlate data obtained from our parallel study ?The Microbiome in VEO-IBD? with the genomic data of Aim 1 to look for changes in the microbiota associated with the defects in the different pathways leading to VEO-IBD. Completion of this project will result in the identification of novel genetic causes of VEO-IBD, providing a fertile source of biologic processes to pursue to better understand the basic mechanisms of disease, with the ultimate goal of translating this knowledge into improved care for children with VEO-IBD through individualized and targeted therapy.